Single Linker FabFv Antibodies and Methods of Producing Same

ABSTRACT

The present disclosure relates to a multi-specific antibody molecule comprising or consisting of three polypeptides: a) a polypeptide chain of formula (I):(Vxx)nVx-Cx-X-V1; and b) a polypeptide chain of formula (II): (Vyy)nVy-Cy c) a polypeptide of formula (III):V2 wherein Vx represents a variable domain, Vxx represents a variable domain, Cx represents a constant region, X represents a linker, V represents a variable domain, Vy represents a variable domain, Vyy represents a variable domain, Cy represents a constant region, V2 represents a variable domain, nindependently represents 0 or 1, wherein the polypeptide chain of formula (I) and the polypeptide chain of formula (II) is aligned such that the constant regions Cx and Cy are paired and the variable domains Vx and Vy are paired to form a binding domain and optionally a disulphide bond is present between V1 and V2, in particular where a disulphide bond is present. The disclosure also extends to pharmaceutical formulation comprising the construct, DNA encoding the constructs and vectors comprising same. The disclosure further extends to a method of expressing the constructs, for example in a host cell and methods for formulating same as a pharmaceutical composition. The disclosure also relates to use of the constructs and formulations in treatment.

This application is a continuation of U.S. patent application Ser. No.14/654,240, filed Jun. 19, 2015, which is a U.S. National Phaseapplication under 35 U.S.C. § 371 of PCT/EP2013/077758, filed Dec. 20,2013, which claims priority from and the benefit of United KingdomApplication No.: 1223276.5, filed on Dec. 21, 2012, the specificationsof which are hereby incorporated by reference in their entireties.

The present disclosure relates to certain multi-specific constructs,pharmaceutical formulations comprising the construct, DNA encoding theconstructs and vectors comprising same. The disclosure also extends to amethod of expressing the constructs, for example in a host cell andmethods for formulating same as a pharmaceutical composition. Thedisclosure also relates to use of the constructs and formulations intreatment.

WO2009/040562 and WO2010/035012 discloses certain bi-specific moleculesuseful as therapeutic agents, known as Fab-Fv or Fab-dsFv respectively.The molecules of this type have good binding affinity for the antigensto which they are specific and no significant occlusion of antigenbinding sites occurs in the format. Whilst a high percentage of theseantibody molecules are expressed as functional monomer there is aproportion that aggregates and from which the monomer needs to bepurified.

The present inventors have re-engineered the molecules concerned toprovide molecules with equivalent functionality, whilst minimisingaggregation at the expression stage and thus substantially increasingthe yield of monomer.

In one embodiment there is provided a multi-specific antibody moleculecomprising or consisting of three polypeptides:

a) a polypeptide chain of formula (I):

(Vxx)_(n)Vx-Cx-X-V₁; and

b) a polypeptide chain of formula (II):

(Vyy)_(n)Vy-C_(y)

c) a polypeptide of formula (III):

V₂

whereinVx represents a variable domain,Vxx represents a variable domain,Cx represents a constant region domain,X represents a linker,V₁ represents a variable domain,Vy represents a variable domain,Vyy represents a variable domain,Cy represents a constant region domain,V₂ represents a variable domain,n independently represents 0 or 1,wherein the polypeptide chain of formula (I) and the polypeptide chainof formula (II) are aligned such that the constant regions Cx and Cy arepaired and the variable domain Vx and Vy are paired to form a bindingdomain and optionally a disulphide bond is present between V₁ and V₂, inparticular where a disulphide bond is present.

In one embodiment Vxx and Vyy are also paired to form a binding domain.

In one embodiment a disulphide bond is present between V₁ and V₂.

In one embodiment there is provided a bi-specific antibody moleculecomprising or consisting of three polypeptides;

a) a heavy chain of formula (Ia):

VH—CH₁—X—V₁; and

b) a light chain of formula (IIa):

VL-C_(L)

c) a polypeptide of formula (III):

V₂

whereinVH represents a heavy chain variable domain,CH₁ represents domain 1 of a heavy chain constant region,X represents a linker,V₁ represents a variable domain,V_(L) represents a light chain variable domain,C_(L) represents a constant region from a light chain,V₂ represents a variable domain,wherein optionally a disulphide bond is present between V₁ and V₂, inparticular where a disulphide bond is present.

In one embodiment there is provided a bi-specific antibody moleculecomprising or consisting of three polypeptides;

a) a heavy chain of formula (Ib):

VH-CH₁; and

b) a light chain of formula (IIb):

VL-C_(L)-X—V₂

c) a polypeptide of formula (III):

V₁

whereinVH represents a heavy chain variable domain,CH₁ represents domain 1 of a heavy chain constant region,X represents a linker,V₁ represents a variable domain,V_(L) represents a light chain variable domain,C_(L) represents a constant region from a light chain,V₂ represents a variable domain,wherein optionally a disulphide bond is present between V₁ and V₂, inparticular where a disulphide bond is present.

In one embodiment a disulphide bond is present between V₁ and V₂.

Advantageously, the present construct minimises the amount ofaggregation seen during expression and maximises the amount of monomerobtained, for example the monomer may be 50%, 60%, 70% or 75% or moresuch as 80 or 90% or more of the protein expressed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows various sequences for single linker Fab-Fv constructsaccording to the invention and comparator constructs Fabdsscfv andFabdsFv. (1A) A26Fab-645dsFv sequences, (1B) Single linkerA26Fab-645dsFv (LC-vL linked) sequences, (1C) Single linkerA26Fab-645dsFv (HC-vH linked) sequences, (1D) A26Fab-645dsscFv (HC-scFv)sequences, (1E) A26Fab-645dsscFv (LC-scFv) sequences, (1F) Fab, Fab-648gand 648g sequences, (1G) 645 CDR, 648 CDR and anti-albumin antibodysequences

FIG. 2 shows SDS-PAGE analysis of the various constructs

FIG. 3 shows size exclusion analysis of various constructs

FIG. 4 shows a diagrammatic representation of various example constructsaccording to the disclosure

FIG. 5 shows transient expression of single linker Fab-dsFvs expressedfrom triple gene plasmids in CHO cells.

FIG. 6 shows SDS-PAGE analysis of various single linker Fab-dsFvsexpressed from triple gene plasmids.

FIG. 7 shows size exclusion analysis of single linker Fab-dsFv expressedfrom a triple gene plasmids.

DETAILED DESCRIPTION OF THE INVENTION

Multi-specific antibody as employed herein refers to an antibodymolecule as described herein which has two or more binding domains, forexample two or three binding domains. In one embodiment the construct isa tri-specific antibody. Tri-specific molecule as employed herein refersto a molecule with three antigen binding sites, which may independentlybind the same or different antigens.

In one embodiment the construct is a bi-specific antibody. Bi-specificmolecule as employed herein refers to a molecule with two antigenbinding sites, which may bind the same or different antigens.

In one embodiment the domains all bind the same antigen, includingbinding the same epitope on the antigen or binding different epitopes onthe antigen.

In one embodiment there are three binding domains and each of the threebinding domains bind different (distinct) antigens.

In one embodiment there are three binding domains and two bindingdomains bind the same antigen, including binding the same epitope ordifferent epitopes on the same antigen, and the third binding domainbinds a different (distinct) antigen.

In one embodiment the present disclosure relates to a bi-specificantibody comprising or consisting of three polypeptide chains.

The multi-specific molecules according to the present disclosure areprovided as a dimer of a heavy and light chain of:

-   -   formula (I) and (II) respectively, wherein the Vx-Cx portion        together with the Vy-Cy portion form a functional Fab or Fab′        fragment, or alternatively    -   formula (Ia) and (IIa), wherein the VH-CH₁ portion together with        the VL-C_(L) form a functional Fab or Fab′ fragment.

In one embodiment the construct of the present disclosure has only twoantigen binding sites.

Antigen binding site as employed herein refers to a portion of themolecule, which comprises a pair of variable regions, in particular acognate pair, that interact specifically with the target antigen.

Specifically as employed herein is intended to refer to a binding sitethat only recognises the antigen to which it is specific or a bindingsite that has significantly higher binding affinity to the antigen towhich is specific compared to affinity to antigens to which it isnon-specific, for example 5, 6, 7, 8, 9, 10 times higher bindingaffinity.

Binding affinity may be measured by standard assay, for example surfaceplasmon resonance, such as BIAcore.

In one embodiment one or more natural or engineered inter chain (i.e.inter light and heavy chain) disulphide bonds are present in thefunctional Fab or Fab′ fragment.

In one embodiment a “natural” disulfide bond is present between a CH₁and C_(L) or corresponding components Cx and Cy in the polypeptidechains of formula (I) and (II). Below references to CH₁ may applyequally to Cx. Below references to C_(L) may apply equally to Cy.

When the C_(L) domain is derived from either Kappa or Lambda the naturalposition for a bond forming cysteine is 214 in human cKappa and cLambda(Kabat numbering 4^(th) edition 1987).

The exact location of the disulfide bond forming cysteine in CH₁ dependson the particular domain actually employed. Thus, for example in humangamma-1 the natural position of the disulfide bond is located atposition 233 (Kabat numbering 4^(th) edition 1987). The position of thebond forming cysteine for other human isotypes such as gamma 2, 3, 4,IgM and IgD are known, for example position 127 for human IgM, IgE,IgG2, IgG3, IgG4 and 128 of the heavy chain of human IgD and IgA2B.

A disulfide bond or bond(s) in the constant region of the molecule maybe in addition to the optional disulfide bond between a variable domainpair V₁ and V₂.

In one embodiment the multi-specific antibody according to thedisclosure has a disulfide bond in a position equivalent orcorresponding to that in the naturally occurring between CH₁ and C_(L).

In one embodiment a constant region comprising CH₁ and a constant regionsuch as C_(L) has a disulfide bond which is in a non-naturally occurringposition. This may be engineered into the molecule by introducingcysteine(s) into the amino acid chain at the position or positionsrequired. This non-natural disulfide bond is in addition to or as analternative to the natural disulfide bond present between CH₁ and C_(L).

Introduction of engineered cysteines can be performed using any methodknown in the art. These methods include, but are not limited to, PCRextension overlap mutagenesis, site-directed mutagenesis or cassettemutagenesis (see, generally, Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbour Laboratory Press, Cold SpringHarbour, N Y, 1989; Ausbel et al., Current Protocols in MolecularBiology, Greene Publishing & Wiley-Interscience, N Y, 1993).Site-directed mutagenesis kits are commercially available, e.g.QuikChange® Site-Directed Mutagenesis kit (Stratagene, La Jolla,Calif.). Cassette mutagenesis can be performed based on Wells et al.,1985, Gene, 34:315-323. Alternatively, mutants can be made by total genesynthesis by annealing, ligation and PCR amplification and cloning ofoverlapping oligonucleotides.

In one embodiment a disulfide bond between CH₁ and C_(L) is completelyabsent, for example the interchain cysteines may be replaced by anotheramino acid, such as serine. Thus there are no inter chain disulphidebonds in the functional Fab fragment of the molecule. Disclosures suchas WO2005/003170, incorporated herein by reference, describe how toprovide Fab fragments without an inter chain disulphide bond.

In one embodiment n is 1 in the polypeptide chain of formula (I).

In one embodiment n is 1 in the polypeptide chain of formula (II).

In one embodiment n is 1 in the polypeptide chain of formula (I) and(II).

In one embodiment n is 0 in the polypeptide chain of formula (I) and(II).

Vxx may be derived from a heavy chain variable region, light chainvariable region or a combination thereof and may comprise an amino acidlinker of 1 to 20 amino acids, for example as described below. In oneembodiment Vxx consists of a variable region, in particular a variableregion derived from a heavy chain. In one embodiment Vxx represents alight chain variable domain. In one embodiment Vxx is a chimericvariable domain, that is to say it comprises components derived from atleast two species, for example a human framework and non-human CDRs. Inone embodiment Vxx is humanised.

Vx may be derived from a heavy chain variable region, light chainvariable region or a combination thereof, in particular a variableregion derived from a heavy chain. In one embodiment Vx represents alight chain variable domain. In one embodiment Vx is a chimeric variabledomain, that is to say it comprises components derived from at least twospecies, for example a human framework and non-human CDRs. In oneembodiment Vx is humanised.

Vx in polypeptides of formula (I) corresponds to VH in polypeptide chain(Ia).

VH represents a variable domain, for example a heavy chain variabledomain. In one embodiment VH represents a heavy chain variable domain.In one embodiment V_(H) is a chimeric variable domain, that is to say itcomprises components derived from at least two species, for example ahuman framework and non-human CDRs. In one embodiment VH is humanised.

V₁ represents a variable domain, for example a heavy chain or lightchain variable domain. In one embodiment V₁ represents a heavy chainvariable domain. In one embodiment V₁ represents a light chain variabledomain. In one embodiment V₁ is a chimeric variable domain, that is tosay it comprises components derived from at least two species, forexample a human framework and non-human CDRs. In one embodiment V₁ ishumanised.

Vyy may be derived from a heavy chain, light chain or a combinationthereof and may comprise an amino acid linker of 1 to 20 amino acids,for example as described below. In one embodiment Vyy consists of avariable region, in particular a variable region derived from a lightchain. In one embodiment Vyy represents a heavy chain variable domain.In one embodiment Vyy is a chimeric variable domain, that is to say itcomprises components derived from at least two species, for example ahuman framework and non-human CDRs. In one embodiment Vyy is humanised.

Vy may be derived from a heavy chain variable region, light chainvariable region or a combination thereof, in particular a variableregion derived from a light chain. In one embodiment Vy represents aheavy chain variable domain. In one embodiment Vy is a chimeric variabledomain, that is to say it comprises components derived from at least twospecies, for example a human framework and non-human CDRs. In oneembodiment Vy is humanised.

Vy in polypeptides of formula (II) corresponds to VL in polypeptides offormula (IIa).

V_(L) represents a variable domain, for example a light chain variabledomain. In one embodiment V_(L) represents a light chain variabledomain. In one embodiment VL is a chimeric variable domain, that is tosay it comprises components derived from at least two species, forexample a human framework and non-human CDRs. In one embodiment V_(L) ishumanised.

V₂ represents a variable domain, for example a heavy chain or lightchain variable domain. In one embodiment V₂ represents a light chainvariable domain. In one embodiment V₂ represents a heavy chain variabledomain. In one embodiment V₂ is a chimeric variable domain, that is tosay it comprises components derived from at least two species, forexample a human framework and non-human CDRs. In one embodiment V₁ ishumanised.

Generally Vxx and Vyy together form an antigen binding domain. In oneembodiment Vxx and Vyy together represent a cognate pair.

Generally Vx and Vy together form an antigen binding domain. In oneembodiment Vx and Vy together represent a cognate pair.

In one embodiment the binding domain formed by VH and VL are specific toa first antigen.

In one embodiment VH and VL form a cognate pair.

Generally V₁ and V₂ together form an antigen binding domain. In oneembodiment V₁ and V₂ together represent a cognate pair.

In one embodiment V₁ and V₂ together form an antigen binding domainspecific for a first antigen (i.e. the two binding domains in themolecule may be specific to the same antigen, for example binding thesame or a different epitope therein).

In one embodiment V₁ and V₂ together are a binding domain for humanserum albumin.

In one embodiment V₁ and V₂ together form an antigen binding domainspecific for a second antigen (i.e. the two binding domains in themolecule are specific to different antigens).

In one embodiment the disulfide bond between V₁ and V₂ is between two ofthe residues listed below (unless the context indicates otherwise Kabatnumbering is employed in the list below). Wherever reference is made toKabat numbering the relevant reference is Kabat et al., 1987, inSequences of Proteins of Immunological Interest, US Department of Healthand Human Services, NIH, USA. In one embodiment the disulfide bond is ina position selected from the group comprising:

-   -   VH37+VL95C see for example Protein Science 6, 781-788 Zhu et al        (1997);    -   VH44+VL100 see for example; Biochemistry 33 5451-5459 Reiter et        al (1994); or Journal of Biological Chemistry Vol. 269 No. 28        pp. 18327-18331 Reiter et al (1994); or Protein Engineering,        vol. 10 no. 12 pp. 1453-1459 Rajagopal et al (1997);    -   VH44+VL105 see for example J Biochem. 118, 825-831 Luo et al        (1995);    -   VH45+VL87 see for example Protein Science 6, 781-788 Zhu et al        (1997);    -   VH55+VL101 see for example FEBS Letters 377 135-139 Young et al        (1995);    -   VH100+VL50 see for example Biochemistry 29 1362-1367 Glockshuber        et al (1990);    -   VH100b+VL49;    -   VH98+VL 46 see for example Protein Science 6, 781-788 Zhu et al        (1997);    -   VH101+VL46;    -   VH105+VL43 see for example; Proc. Natl. Acad. Sci. USA Vol. 90        pp. 7538-7542 Brinkmann et al (1993); or Proteins 19, 35-47 Jung        et al (1994),    -   VH106+VL57 see for example FEBS Letters 377 135-139 Young et        al (1995) and a position corresponding thereto in variable        region pair located in the molecule.

The amino acid pairs listed above are in the positions conducive toreplacement by cysteines such that disulfide bonds can be formed.Cysteines can be engineered into these desired positions by knowntechniques. In one embodiment therefore an engineered cysteine accordingto the present invention refers to where the naturally occurring residueat a given amino acid position has been replaced with a cysteineresidue.

Introduction of engineered cysteines can be performed using any methodknown in the art. These methods include, but are not limited to, PCRextension overlap mutagenesis, site-directed mutagenesis or cassettemutagenesis (see, generally, Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbour Laboratory Press, Cold SpringHarbour, N Y, 1989; Ausbel et al., Current Protocols in MolecularBiology, Greene Publishing & Wiley-Interscience, N Y, 1993).Site-directed mutagenesis kits are commercially available, e.g.QuikChange® Site-Directed Mutagenesis kit (Stratagen, La. Jolla,Calif.). Cassette mutagenesis can be performed based on Wells et al.,1985, Gene, 34:315-323. Alternatively, mutants can be made by total genesynthesis by annealing, ligation and PCR amplification and cloning ofoverlapping oligonucleotides.

Accordingly in one embodiment a variable domain pair (V₁/V₂) of thepresent invention may be linked by a disulfide bond between two cysteineresidues, one in V₁ and one in V₂, wherein the position of the pair ofcysteine residues is selected from the group consisting of VH37 andVL95, VH44 and VL100, VH44 and VL105, VH45 and VL87, VH100 and VL50,VH100b and VL49, VH98 and VL46, VH101 and VL46, VH105 and VL43 and VH106and VL57.

In one embodiment a variable domain pair (V₁/V₂) of the presentinvention may be linked by a disulfide bond between two cysteineresidues, one in V₁ and one in V₂, which are outside of the CDRs whereinthe position of the pair of cysteine residues is selected from the groupconsisting of VH37 and VL95, VH44 and VL100, VH44 and VL105, VH45 andVL87, VH100 and VL50, VH98 and VL46, VH105 and VL43 and VH106 and VL57.

In one embodiment V₁ is a heavy chain variable domain and V₂ is a lightchain variable domain and V₁ and V₂ are linked by a disulphide bondbetween two engineered cysteine residues, one at position VH44 of V₁ andthe other at VL100 of V₂.

In one embodiment VH and V₁ are variable regions which are both from aheavy chain(s) or a light chain(s), in particular are both derived fromtwo distinct heavy chain variable regions.

In one embodiment VL and V₂ are variable regions which are both from aheavy chain(s) or a light chain(s), in particular are both derived fromtwo distinct light chain variable regions.

Cognate pair as employed herein refers to a pair of variable domainsfrom a single antibody, which was generated in vivo, i.e. the naturallyoccurring pairing of the variable domains isolated from a host. Acognate pair is therefore a VH and VL pair. In one example the cognatepair bind the antigen co-operatively.

Variable region as employed herein refers to the region in an antibodychain comprising the CDRs and a suitable framework.

Variable regions for use in the present disclosure will generally bederived from an antibody, which may be generated by any method known inthe art.

Derived from as employed herein refers to the fact that the sequenceemployed or a sequence highly similar to the sequence employed wasobtained from the original genetic material, such as the light or heavychain of an antibody.

Highly similar as employed herein is intended to refer to an amino acidsequence which over its full length is 95% similar or more, such as 96,97, 98 or 99% similar.

Antibodies generated against the antigen polypeptide may be obtained,where immunisation of an animal is necessary, by administering thepolypeptides to an animal, preferably a non-human animal, usingwell-known and routine protocols, see for example Handbook ofExperimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell ScientificPublishers, Oxford, England, 1986). Many warm-blooded animals, such asrabbits, mice, rats, sheep, cows, camels or pigs may be immunized.However, mice, rabbits, pigs and rats are generally most suitable.

Monoclonal antibodies may be prepared by any method known in the artsuch as the hybridoma technique (Kohler & Milstein, 1975, Nature,256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today, 4:72) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

Antibodies may also be generated using single lymphocyte antibodymethods by cloning and expressing immunoglobulin variable region cDNAsgenerated from single lymphocytes selected for the production ofspecific antibodies by, for example, the methods described by Babcook,J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481;WO92/02551; WO2004/051268 and WO2004/106377.

The antibodies for use in the present invention can also be generatedusing various phage display methods known in the art and include thosedisclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50),Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough etal. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 1879-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) andWO90/02809; WO91/10737; WO92/01047; WO92/18619; WO93/11236; WO95/15982;WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;5,516,637; 5,780,225; 5,658,727; 5,733,743; 5,969,108, andWO20011/30305.

In one embodiment the bi-specific molecules according to the disclosureare humanised.

Humanised (which include CDR-grafted antibodies) as employed hereinrefers to molecules having one or more complementarity determiningregions (CDRs) from a non-human species and a framework region from ahuman immunoglobulin molecule (see, e.g. U.S. Pat. No. 5,585,089;WO91/09967). It will be appreciated that it may only be necessary totransfer the specificity determining residues of the CDRs rather thanthe entire CDR (see for example, Kashmiri et al., 2005, Methods, 36,25-34). Humanised antibodies may optionally further comprise one or moreframework residues derived from the non-human species from which theCDRs were derived.

As used herein, the term ‘humanised antibody molecule’ refers to anantibody molecule wherein the heavy and/or light chain contains one ormore CDRs (including, if desired, one or more modified CDRs) from adonor antibody (e.g. a murine monoclonal antibody) grafted into a heavyand/or light chain variable region framework of an acceptor antibody(e.g. a human antibody). For a review, see Vaughan et al, NatureBiotechnology, 16, 535-539, 1998. In one embodiment rather than theentire CDR being transferred, only one or more of the specificitydetermining residues from any one of the CDRs described herein above aretransferred to the human antibody framework (see for example, Kashmiriet al., 2005, Methods, 36, 25-34). In one embodiment only thespecificity determining residues from one or more of the CDRs describedherein above are transferred to the human antibody framework. In anotherembodiment only the specificity determining residues from each of theCDRs described herein above are transferred to the human antibodyframework.

When the CDRs or specificity determining residues are grafted, anyappropriate acceptor variable region framework sequence may be usedhaving regard to the class/type of the donor antibody from which theCDRs are derived, including mouse, primate and human framework regions.Suitably, the humanised antibody according to the present invention hasa variable domain comprising human acceptor framework regions as well asone or more of the CDRs provided herein.

Examples of human frameworks which can be used in the present inventionare KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). Forexample, KOL and NEWM can be used for the heavy chain, REI can be usedfor the light chain and EU, LAY and POM can be used for both the heavychain and the light chain. Alternatively, human germline sequences maybe used; these are available at: In a humanised antibody of the presentinvention, the acceptor heavy and light chains do not necessarily needto be derived from the same antibody and may, if desired, comprisecomposite chains having framework regions derived from different chains.

The framework regions need not have exactly the same sequence as thoseof the acceptor antibody. For instance, unusual residues may be changedto more frequently-occurring residues for that acceptor chain class ortype. Alternatively, selected residues in the acceptor framework regionsmay be changed so that they correspond to the residue found at the sameposition in the donor antibody (see Reichmann et al., 1998, Nature, 332,323-324). Such changes should be kept to the minimum necessary torecover the affinity of the donor antibody. A protocol for selectingresidues in the acceptor framework regions which may need to be changedis set forth in WO91/09967.

In one embodiment the bi-specific antibodies of the present disclosureare fully human, in particular one or more of the variable domains arefully human.

Fully human molecules are those in which the variable regions and theconstant regions (where present) of both the heavy and the light chainsare all of human origin, or substantially identical to sequences ofhuman origin, not necessarily from the same antibody. Examples of fullyhuman antibodies may include antibodies produced, for example by thephage display methods described above and antibodies produced by mice inwhich the murine immunoglobulin variable and optionally the constantregion genes have been replaced by their human counterparts eg. asdescribed in general terms in EP0546073 B1, U.S. Pat. Nos. 5,545,806,5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, EP 0438474 andEP0463151.

Cx is a constant domain from a light or heavy chain, in particular aheavy chain.

Cx in polypeptide of formula (I) corresponds to CH₁ in polypeptides offormula (Ia). In one embodiment Cx is equivalent to CH₁.

In one embodiment the CH₁ domain is a naturally occurring domain 1 froma heavy chain or a derivative thereof. In one embodiment the CH fragmentconsists of a CH₁ domain.

Cy is a constant domain from a light or heavy chain, in particular alight chain.

Cy in polypeptide of formula (II) corresponds to CL in polypeptides offormula (IIa). In one embodiment Cy is equivalent to CL.

In one embodiment the C_(L) fragment, in the light chain, is a constantkappa sequence or a constant lambda sequence or a derivative thereof.

A derivative of a naturally occurring domain as employed herein isintended to refer to where one, two, three, four or five amino acids ina naturally occurring sequence have been replaced or deleted, forexample to optimize the properties of the domain such as by eliminatingundesirable properties but wherein the characterizing feature(s) of thedomain is/are retained.

In one embodiment X is a linker for example a suitable peptide forconnecting the portions CH₁ and V₁.

In one embodiment X is a linker for example a suitable peptide forconnecting the portions C_(L) and V₂.

In one embodiment the peptide linker is 50 amino acids in length orless, for example 20 amino acids or less.

In one embodiment the linker is selected from a sequence shown insequence 13 to 77.

In one embodiment the linker is selected from a sequence shown in SEQ IDNO: 103 or SEQ ID NO:104.

TABLE 1 Hinge linker sequences SEQ ID NO: SEQUENCE 13 DKTHTCAA 14DKTHTCPPCPA 15 DKTHTCPPCPATCPPCPA 16 DKTHTCPPCPATCPPCPATCPPCPA 17DKTHTCPPCPAGKPTLYNSLVMSDTAGTCY 18 DKTHTCPPCPAGKPTHVNVSVVMAEVDGTCY 19DKTHTCCVECPPCPA 20 DKTHTCPRCPEPKSCDTPPPCPRCPA 21 DKTHTCPSCPA

TABLE 2 Flexible linker sequences SEQ ID NO: SEQUENCE 22 SGGGGSE 23DKTHTS 24 (S)GGGGS 25 (S)GGGGSGGGGS 26 (S)GGGGSGGGGSGGGGS 27(S)GGGGSGGGGSGGGGSGGGGS 28 (S)GGGGSGGGGSGGGGSGGGGSGGGGS 29 AAAGSG-GASAS30 AAAGSG-XGGGS-GASAS 31 AAAGSG-XGGGSXGGGS-GASAS 32AAAGSG-XGGGSXGGGSXGGGS-GASAS 33 AAAGSG-XGGGSXGGGSXGGGSXGGGS-GASAS 34AAAGSG-XS-GASAS 35 PGGNRGTTTTRRPATTTGSSPGPTQSHY 36 ATTTGSSPGPT 37 ATTTGS— GS 38 EPSGPISTINSPPSKESHKSP 39 GTVAAPSVFIFPPSD 40 GGGGIAPSMVGGGGS 41GGGGKVEGAGGGGGS 42 GGGGSMKSHDGGGGS 43 GGGGNLITIVGGGGS 44 GGGGVVPSLPGGGGS45 GGEKSIPGGGGS 46 RPLSYRPPFPFGFPSVRP 47 YPRSIYIRRRHPSPSLTT 48TPSHLSHILPSFGLPTFN 49 RPVSPFTFPRLSNSWLPA 50 SPAAHFPRSIPRPGPIRT 51APGPSAPSHRSLPSRAFG 52 PRNSIHFLHPLLVAPLGA 53 MPSLSGVLQVRYLSPPDL 54SPQYPSPLTLTLPPHPSL 55 NPSLNPPSYLHRAPSRIS 56 LPWRTSLLPSLPLRRRP 57PPLFAKGPVGLLSRSFPP 58 VPPAPVVSLRSAHARPPY 59 LRPTPPRVRSYTCCPTP- 60PNVAHVLPLLTVPWDNLR 61 CNPLLPLCARSPAVRTFP

(S) is optional in sequences 24 to 28.

Examples of rigid linkers include the peptide sequences GAPAPAAPAPA (SEQID NO: 62), PPPP (SEQ ID NO: 63) and PPP.

In one embodiment the peptide linker is an albumin binding peptide.

Examples of albumin binding peptides are provided in WO2007/106120 andinclude:

TABLE 3 SEQ ID NO: SEQUENCE 64 DLCLRDWGCLW 65 DICLPRWGCLW 66MEDICLPRWGCLWGD 67 QRLMEDICLPRWGCLWEDDE 68 QGLIGDICLPRWGCLWGRSV 69QGLIGDICLPRWGCLWGRSVK 70 EDICLPRWGCLWEDD 71 RLMEDICLPRWGCLWEDD 72MEDICLPRWGCLWEDD 73 MEDICLPRWGCLWED 74 RLMEDICLARWGCLWEDD 75EVRSFCTRWPAEKSCKPLRG 76 RAPESFVCYWETICFERSEQ 77 EMCYFPGICWM

Advantageously use of albumin binding peptides as a linker may increasethe half-life of the bi-specific antibody molecule.

For the avoidance of doubt V₂ is still present in the antibody moleculeand is retained therein by virtue of pairing with V₁ including where adisulphide bond is present between V₁ and V₂.

In one embodiment the bi-specific antibody molecules of the disclosureare capable of selectively binding two different antigens of interest.

In one embodiment, an antigen of interest bound by Vxx/Vyy, Vx/Vy, VH/VLand V₁/V₂ are independently selected from a cell-associated protein, forexample a cell surface protein on cells such as bacterial cells, yeastcells, T-cells, endothelial cells or tumour cells, and a solubleprotein.

Antigens of interest may also be any medically relevant protein such asthose proteins upregulated during disease or infection, for examplereceptors and/or their corresponding ligands. Particular examples ofcell surface proteins include adhesion molecules, for example integrinssuch as 11 integrins e.g. VLA-4, E-selectin, P selectin or L-selectin,CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD20, CD23,CD25, CD33, CD38, CD40, CD45, CDW52, CD69, CD134 (OX40), ICOS, BCMP7,CD137, CD27L, CDCP1, DPCR1, DPCR1, dudulin2, FLJ20584, FLJ40787, HEK2,KIAA0634, KIAA0659, KIAA1246, KIAA1455, LTBP2, LTK, MAL2, MRP2,nectin-like2, NKCC1, PTK7, RAIG1, TCAM1, SC6, BCMP101, BCMP84, BCMP 11,DTD, carcinoembryonic antigen (CEA), human milk fat globulin (HMFG1 and2), MHC Class I and MHC Class II antigens, and VEGF, and whereappropriate, receptors thereof.

Soluble antigens include interleukins such as IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-8, IL-12, IL-16 or IL-17, IL-23, viral antigens forexample respiratory syncytial virus or cytomegalovirus antigens,immunoglobulins, such as IgE, interferons such as interferon α,interferon β or interferon γ, tumour necrosis factor-α, tumor necrosisfactor-β, colony stimulating factors such as G-CSF or GM-CSF, andplatelet derived growth factors such as PDGF-α, and PDGF-β and whereappropriate receptors thereof. Other antigens include bacterial cellsurface antigens, bacterial toxins, viruses such as influenza, EBV,HepA, B and C, bioterrorism agents, radionuclides and heavy metals, andsnake and spider venoms and toxins.

In one embodiment, the antibody fusion protein of the invention may beused to functionally alter the activity of the antigen of interest. Forexample, the antibody fusion protein may neutralize, antagonize oragonise the activity of said antigen, directly or indirectly.

In one embodiment the antigen of interest bound by VH and VL is OX40. Inone embodiment the Vx-Cx or VHCH1 portion of the multi-specific antibodyhas the sequence given in SEQ ID NO:3. In one embodiment the Vy-Cy orVL-CL portion of the multi-specific antibody has the sequence given inSEQ ID NO:7.

In one embodiment, an antigen of interest bound by VH/VL or V₁/V₂provides the ability to recruit effector functions, such as complementpathway activation and/or effector cell recruitment.

The recruitment of effector function may be direct in that effectorfunction is associated with a cell, said cell bearing a recruitmentmolecule on its surface. Indirect recruitment may occur when binding ofa binding domain (such as V₁/V₂) in the molecule according to presentdisclosure to a recruitment polypeptide causes release of, for example,a factor which in turn may directly or indirectly recruit effectorfunction, or may be via activation of a signalling pathway. Examplesinclude TNFα, IL2, IL6, IL8, IL17, IFNγ, histamine, C1q, opsonin andother members of the classical and alternative complement activationcascades, such as C2, C4, C3-convertase, and C5 to C9.

As used herein, ‘a recruitment polypeptide’ includes a FcγR such asFcγRI, FcγRII and FcγRIII, a complement pathway protein such as, butwithout limitation, C1q and C3, a CD marker protein (Cluster ofDifferentiation marker) such as, but without limitation, CD68, CD115,CD16, CD80, CD83, CD86, CD56, CD64, CD3, CD4, CD8, CD28, CD45, CD19,CD20 and CD22. Further recruitment polypeptides which are CD markerproteins include CD1, CD1d, CD2, CD5, CD8, CD9, CD10, CD11, CD11a,CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22,CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34,CD35, CD36, CD37, CD38, CD40, CD43, CD44, CD45, CD46, CD49, CD49a,CD49b, CD49c, CD49d, CD52, CD53, CD54, CD55, CD56, CD58, CD59, CD61,CD62, D62E, CD62L, CD62P, CD63, CD64, CD66e, CD68, CD70, CD71, CD72,CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD88, CD89, CD90, CD94,CD95, CD98, CD106, CD114, CD116, CD117, CD118, CD120, CD122, CD130,CD131, CD132, CD133, CD134, CD135, CD137, CD138, CD141, CD142, CD143,CD146, CD147, CD151, CD152, CD153, CD154, CD155, CD162, CD164, CD169,CD184, CD206, CD209, CD257, CD278, CD281, CD282, CD283 and CD304, or afragment of any thereof which retains the ability to recruitcell-mediated effector function either directly or indirectly. Arecruitment polypeptide also includes immunoglobulin molecules such asIgG1, IgG2, IgG3, IgG4, IgE and IgA which possess effector function.

In one embodiment, a binding domain (such as V₁/V₂) in the moleculeaccording to the present disclosure has specificity is a complementpathway protein, with C1q being particularly preferred.

Further, molecules of the present invention may be used to chelateradionuclides by virtue of a single domain antibody which binds to anuclide chelator protein. Such fusion proteins are of use in imaging orradionuclide targeting approaches to therapy.

In one embodiment, one binding domain in a molecule according to thedisclosure (such as V₁/V₂) has specificity is a CD marker protein, withCD68, CD80, CD86, CD64, CD3, CD4, CD8 CD45, CD16 and CD35 beingparticularly preferred.

In one embodiment a binding domain within a molecule according to thedisclosure (such as V₁/V₂) has specificity for a serum carrier protein,a circulating immunoglobulin molecule, or CD35/CR1, for example forproviding an extended half-life to the antibody fragment withspecificity for said antigen of interest by binding to said serumcarrier protein, circulating immunoglobulin molecule or CD35/CR1.

As used herein, ‘serum carrier proteins’ include thyroxine-bindingprotein, transthyretin, al-acid glycoprotein, transferrin, fibrinogenand albumin, or a fragment of any thereof.

As used herein, a ‘circulating immunoglobulin molecule’ includes IgG1,IgG2, IgG3, IgG4, sIgA, IgM and IgD, or a fragment of any thereof.

CD35/CR1 is a protein present on red blood cells which have a half-lifeof 36 days (normal range of 28 to 47 days; Lanaro et al., 1971, Cancer,28(3):658-661).

In one embodiment, the protein for which V₁/V₂ has specificity is aserum carrier protein, such as a human serum carrier. In a mostpreferred embodiment, the serum carrier protein is human serum albumin.

Albumin binding variable regions and CDRs are disclosed in constructsshown in FIG. 1.

Thus in one embodiment there is provided a bi-specific antibody moleculecomprising or consisting of three polypeptides;

a) a heavy chain of formula (Ia):

VH-CH₁—X—V₁; and

b) a light chain of formula (IIa):

VL-C_(L)

c) a polypeptide of formula (III):

V₂

whereinV_(H) represents a heavy chain variable domain,CH₁ represents domain 1 of a heavy chain constant region,X represents a linker,V₁ represents a variable domain,V_(L) represents a light chain variable domain,C_(L) represents a constant region from a light chain,V₂ represents a variable region,wherein V_(H) and V_(L) are a cognate pair of variable regions alignedto form a binding domain and V₁ and V₂ are a cognate pair of variableregions aligned to form a binding domain optionally a disulphide bondthere-between, for example wherein V₁ and V₂ are capable of bindingalbumin in vivo, in particular human serum albumin.

In one embodiment V1 or V2 comprise a sequence selected from the groupconsisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:83 and SEQ ID NO:86.In one embodiment V1 has the sequence given in SEQ ID NO:4 and V2 hasthe sequence given in SEQ ID NO:8. In one embodiment V1 has the sequencegiven in SEQ ID NO:86 and V2 has the sequence given in SEQ ID NO:83.

In one example V1 or V2, in particular V1, is a VH domain comprising thesequence given in SEQ ID NO:87 for CDRH-1, the sequence given in SEQ IDNO:88 for CDRH2 and the sequence given in SEQ ID NO:89 for CDRH-3. Inone example V1 or V2, in particular V1, is a VH domain comprising thesequence given in SEQ ID NO:93 for CDRH-1, the sequence given in SEQ IDNO:94 for CDRH2 and the sequence given in SEQ ID NO:95 for CDRH-3.

In one embodiment V1 or V2, in particular V2, is a VL domain comprisingthe sequence given in SEQ ID NO:90 for CDRL-1, the sequence given in SEQID NO:91 for CDRL2 and the sequence given in SEQ ID NO:92 for CDRL-3. Inone embodiment V1 or V2, in particular V2, is a VL domain comprising thesequence given in SEQ ID NO:96 for CDRL-1, the sequence given in SEQ IDNO:97 for CDRL2 and the sequence given in SEQ ID NO:98 for CDRL-3.

In one example V1 or V2, in particular V1, is a VH domain comprising thesequence given in SEQ ID NO:4, SEQ ID NO: 86, SEQ NO:99 or SEQ ID NO:100.

In one example V1 or V2, in particular V2, is a VL domain comprising thesequence given in SEQ ID NO: 8, SEQ ID NO: 83, SEQ NO: 101 or SEQ ID NO:102.

In one example V1 is a VH domain comprising the sequence given in SEQNO:99 and V2 is a VL domain comprising the sequence given in SEQ NO:101.

In one example V1 is a VH domain comprising the sequence given in SEQNO: 100 and V2 is a VL domain comprising the sequence given in SEQ NO:102.

In one example polypeptide Ia has the sequence given in SEQ ID NO:6.

In one example polypeptide IIa has the sequence given in SEQ ID NO:7.

In one example polypeptide Ib has the sequence given in SEQ ID NO:3.

In one example polypeptide IIb has the sequence given in SEQ ID NO:5.

In one example polypeptide Ia has the sequence given in SEQ ID NO:6,polypeptide IIa has the sequence given in SEQ ID NO:7 and V₂ has thesequence given in SEQ ID NO:8.

In one example polypeptide Ib has the sequence given in SEQ ID NO:3,polypeptide IIb has the sequence given in SEQ ID NO:5 and V₁ has thesequence given in SEQ ID NO:4.

The invention also provides sequences which are 80%, 90%, 91%, 92%, 93%94%, 95% 96%, 97%, 98% or 99% similar to a sequence or antibodydisclosed herein.

“Identity”, as used herein, indicates that at any particular position inthe aligned sequences, the amino acid residue is identical between thesequences. “Similarity”, as used herein, indicates that, at anyparticular position in the aligned sequences, the amino acid residue isof a similar type between the sequences. For example, leucine may besubstituted for isoleucine or valine. Other amino acids which can oftenbe substituted for one another include but are not limited to:

-   -   phenylalanine, tyrosine and tryptophan (amino acids having        aromatic side chains);    -   lysine, arginine and histidine (amino acids having basic side        chains);    -   aspartate and glutamate (amino acids having acidic side chains);    -   asparagine and glutamine (amino acids having amide side chains);        and    -   cysteine and methionine (amino acids having sulphur-containing        side chains). Degrees of identity and similarity can be readily        calculated (Computational Molecular Biology, Lesk, A. M., ed.,        Oxford University Press, New York, 1988; Biocomputing.        Informatics and Genome Projects, Smith, D. W., ed., Academic        Press, New York, 1993; Computer Analysis of Sequence Data, Part        1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New        Jersey, 1994; Sequence Analysis in Molecular Biology, von        Heinje, G., Academic Press, 1987, Sequence Analysis Primer,        Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,        1991, the BLAST™ software available from NCBI (Altschul, S. F.        et al., 1990, J. Mol. Biol. 215:403-410; Gish, W. &        States, D. J. 1993, Nature Genet. 3:266-272. Madden, T. L. et        al., 1996, Meth. Enzymol. 266:131-141; Altschul, S. F. et al.,        1997, Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, T. L.        1997, Genome Res. 7:649-656,).

In an alternative aspect the present invention provides a Fab or Fab′fragment linked to a disulphide stabilised scFv, wherein the disulphidestabilised scFv (dsscFv) is linked to the C-terminus of the heavy or thelight chain of the Fab or Fab′ fragment directly or via a linker such asa linker described herein. Preferably the Fab-dsscFv fusion protein isbi-specific. In one example the dsscFv binds a serum carrier proteinsuch as human serum albumin. In one example the dsscFv is linked toC-terminus of the heavy chain of the Fab or Fab′ fragment by the linkergiven in SEQ ID NO:78. In one example the dsscFv is linked to theC-terminus of the light chain of the Fab or Fab′ fragment by the linkergiven in SEQ ID NO: 103. In one example the heavy chain of theFab-dsscFv has the sequence given in SEQ ID NO:9 and the light chain hasthe sequence given in SEQ ID NO: 10. In one example the heavy chain ofthe Fab-dsscFv has the sequence given in SEQ ID NO: 11 and the lightchain has the sequence given in SEQ ID NO: 12.

In one embodiment the bi-specific antibody molecules of the presentdisclosure are processed to provide improved affinity for a targetantigen or antigens. Such variants can be obtained by a number ofaffinity maturation protocols including mutating the CDRs (Yang et al.,J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al.,Bio/Technology, 10, 779-783, 1992), use of mutator strains of E. coli(Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Pattenet al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display(Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexual PCR(Crameri et al., Nature, 391, 288-291, 1998). Vaughan et al. (supra)discusses these methods of affinity maturation.

Improved affinity as employed herein in this context refers to animprovement over the starting molecule.

If desired an antibody for use in the present invention may beconjugated to one or more effector molecule(s). It will be appreciatedthat the effector molecule may comprise a single effector molecule ortwo or more such molecules so linked as to form a single moiety that canbe attached to the antibodies of the present invention. Where it isdesired to obtain an antibody fragment linked to an effector molecule,this may be prepared by standard chemical or recombinant DNA proceduresin which the antibody fragment is linked either directly or via acoupling agent to the effector molecule. Techniques for conjugating sucheffector molecules to antibodies are well known in the art (see,Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al.,eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123).Particular chemical procedures include, for example, those described inWO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and WO03031581.Alternatively, where the effector molecule is a protein or polypeptidethe linkage may be achieved using recombinant DNA procedures, forexample as described in WO 86/01533 and EP0392745.

The term effector molecule as used herein includes, for example,biologically active proteins, for example enzymes, other antibody orantibody fragments, synthetic or naturally occurring polymers, nucleicacids and fragments thereof e.g. DNA, RNA and fragments thereof,radionuclides, particularly radioiodide, radioisotopes, chelated metals,nanoparticles and reporter groups such as fluorescent compounds orcompounds which may be detected by NMR or ESR spectroscopy.

Other effector molecules may include chelated radionuclides such as111In and 90Y, Lu177, Bismuth213, Californium252, Iridium 192 andTungsten 188/Rhenium 188; or drugs such as but not limited to,alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

Other effector molecules include proteins, peptides and enzymes. Enzymesof interest include, but are not limited to, proteolytic enzymes,hydrolases, lyases, isomerases, transferases. Proteins, polypeptides andpeptides of interest include, but are not limited to, immunoglobulins,toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheriatoxin, a protein such as insulin, tumour necrosis factor, α-interferon,β-interferon, nerve growth factor, platelet derived growth factor ortissue plasminogen activator, a thrombotic agent or an anti-angiogenicagent, e.g. angiostatin or endostatin, or, a biological responsemodifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2(IL-2), granulocyte macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF)or other growth factor and immunoglobulins.

Other effector molecules may include detectable substances useful forexample in diagnosis. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive nuclides, positronemitting metals (for use in positron emission tomography), andnonradioactive paramagnetic metal ions. See generally U.S. Pat. No.4,741,900 for metal ions which can be conjugated to antibodies for useas diagnostics. Suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;suitable prosthetic groups include streptavidin, avidin and biotin;suitable fluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride and phycoerythrin; suitable luminescentmaterials include luminol; suitable bioluminescent materials includeluciferase, luciferin, and aequorin; and suitable radioactive nuclidesinclude 125I, 131I, 111In and 99Tc.

In another example the effector molecule may increase the half-life ofthe antibody in vivo, and/or reduce immunogenicity of the antibodyand/or enhance the delivery of an antibody across an epithelial barrierto the immune system. Examples of suitable effector molecules of thistype include polymers, albumin, albumin binding proteins or albuminbinding compounds such as those described in WO05/117984.

Where the effector molecule is a polymer it may, in general, be asynthetic or a naturally occurring polymer, for example an optionallysubstituted straight or branched chain polyalkylene, polyalkenylene orpolyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g.a homo- or hetero-polysaccharide.

Specific optional substituents which may be present on theabove-mentioned synthetic polymers include one or more hydroxy, methylor methoxy groups.

Specific examples of synthetic polymers include optionally substitutedstraight or branched chain poly(ethyleneglycol), poly(propyleneglycol)poly(vinylalcohol) or derivatives thereof, especially optionallysubstituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) orderivatives thereof.

Specific naturally occurring polymers include lactose, amylose, dextran,glycogen or derivatives thereof. “Derivatives” as used herein isintended to include reactive derivatives, for example thiol-selectivereactive groups such as maleimides and the like. The reactive group maybe linked directly or through a linker segment to the polymer. It willbe appreciated that the residue of such a group will in some instancesform part of the product as the linking group between the antibodyfragment and the polymer.

The size of the polymer may be varied as desired, but will generally bein an average molecular weight range from 500 Da to 50000 Da, forexample from 5000 to 40000 Da such as from 20000 to 40000 Da. Thepolymer size may in particular be selected on the basis of the intendeduse of the product for example ability to localize to certain tissuessuch as tumors or extend circulating half-life (for review see Chapman,2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example,where the product is intended to leave the circulation and penetratetissue, for example for use in the treatment of a tumour, it may beadvantageous to use a small molecular weight polymer, for example with amolecular weight of around 5000 Da. For applications where the productremains in the circulation, it may be advantageous to use a highermolecular weight polymer, for example having a molecular weight in therange from 20000 Da to 40000 Da.

Suitable polymers include a polyalkylene polymer, such as apoly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or aderivative thereof, and especially with a molecular weight in the rangefrom about 15000 Da to about 40000 Da.

In one example antibodies for use in the present invention are attachedto poly(ethyleneglycol) (PEG) moieties. In one particular example theantibody is an antibody fragment and the PEG molecules may be attachedthrough any available amino acid side-chain or terminal amino acidfunctional group located in the antibody fragment, for example any freeamino, imino, thiol, hydroxyl or carboxyl group. Such amino acids mayoccur naturally in the antibody fragment or may be engineered into thefragment using recombinant DNA methods (see for example U.S. Pat. Nos.5,219,996; 5,667,425; WO98/25971, WO2008/038024). In one example theantibody molecule of the present invention is a modified Fab fragmentwherein the modification is the addition to the C-terminal end of itsheavy chain one or more amino acids to allow the attachment of aneffector molecule. Suitably, the additional amino acids form a modifiedhinge region containing one or more cysteine residues to which theeffector molecule may be attached. Multiple sites can be used to attachtwo or more PEG molecules.

Suitably PEG molecules are covalently linked through a thiol group of atleast one cysteine residue located in the antibody fragment. Eachpolymer molecule attached to the modified antibody fragment may becovalently linked to the sulphur atom of a cysteine residue located inthe fragment. The covalent linkage will generally be a disulphide bondor, in particular, a sulphur-carbon bond. Where a thiol group is used asthe point of attachment appropriately activated effector molecules, forexample thiol selective derivatives such as maleimides and cysteinederivatives may be used. An activated polymer may be used as thestarting material in the preparation of polymer-modified antibodyfragments as described above. The activated polymer may be any polymercontaining a thiol reactive group such as an α-halocarboxylic acid orester, e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone ora disulphide. Such starting materials may be obtained commercially (forexample from Nektar, formerly Shearwater Polymers Inc., Huntsville,Ala., USA) or may be prepared from commercially available startingmaterials using conventional chemical procedures. Particular PEGmolecules include 20K methoxy-PEG-amine (obtainable from Nektar,formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA(obtainable from Nektar, formerly Shearwater).

In one embodiment, a Fab or Fab′ in the molecule is PEGylated, i.e. hasPEG (poly(ethyleneglycol)) covalently attached thereto, e.g. accordingto the method disclosed in EP 0948544 or EP1090037 [see also“Poly(ethyleneglycol) Chemistry, Biotechnical and BiomedicalApplications”, 1992, J. Milton Harris (ed), Plenum Press, New York,“Poly(ethyleneglycol) Chemistry and Biological Applications”, 1997, J.Milton Harris and S. Zalipsky (eds), American Chemical Society,Washington D.C. and “Bioconjugation Protein Coupling Techniques for theBiomedical Sciences”, 1998, M. Aslam and A. Dent, Grove Publishers, NewYork; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002,54:531-545]. In one example PEG is attached to a cysteine in the hingeregion. In one example, a PEG modified Fab fragment has a maleimidegroup covalently linked to a single thiol group in a modified hingeregion. A lysine residue may be covalently linked to the maleimide groupand to each of the amine groups on the lysine residue may be attached amethoxypoly(ethyleneglycol) polymer having a molecular weight ofapproximately 20,000 Da. The total molecular weight of the PEG attachedto the Fab fragment may therefore be approximately 40,000 Da.

Particular PEG molecules include 2-[3-(N-maleimido)propionamido]ethylamide of N,N′-bis(methoxypoly(ethylene glycol) MW 20,000) modifiedlysine, also known as PEG2MAL40K (obtainable from Nektar, formerlyShearwater).

Alternative sources of PEG linkers include NOF who supply GL2-400MA2(wherein m in the structure below is 5) and GL2-400MA (where m is 2) andn is approximately 450:

-   -   m is 2 or 6        That is to say each PEG is about 20,000 Da.        Further alternative PEG effector molecules of the following        type:

are available from Dr Reddy, NOF and Jenkem.

In one embodiment there is provided an antibody which is PEGylated (forexample with a PEG described herein), attached through a cysteine aminoacid residue at or about amino acid 226 in the chain, for example aminoacid 226 of the heavy chain (by sequential numbering).

In one embodiment there is provided a polynucleotide sequence encoding amolecule of the present disclosure, such as a DNA sequence.

In one embodiment there is provided a polynucleotide sequence encodingone or more, such as two or more polypeptide components of a molecule ofthe present disclosure, for example

a polypeptide chain of formula (I):

(Vxx)_(n)Vx-Cx-X-V₁

a polypeptide chain of formula (II):

(Vyy)_(n)Vy-C_(y)

or a polypeptide of formula (III):

V₂

whereinVx represents a variable domain,Vxx represents a variable domain,Cx represents a constant region domain,X represents a linker,V₁ represents a variable domain,Vy represents a variable domain,Vyy represents a variable domain,Cy represents a constant region domain,V₂ represents a variable domain,

-   -   n independently represents 0 or 1.

In one embodiment the polynucleotide, such as the DNA is comprised in avector.

General methods by which the vectors may be constructed, transfectionmethods and culture methods are well known to those skilled in the art.In this respect, reference is made to “Current Protocols in MolecularBiology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and theManiatis Manual produced by Cold Spring Harbor Publishing.

Also provided is a host cell comprising one or more cloning orexpression vectors comprising one or more DNA sequences encoding anantibody of the present invention. Any suitable host cell/vector systemmay be used for expression of the DNA sequences encoding the antibodymolecule of the present invention.

Bacterial, for example E. coli, and other microbial systems may be usedor eukaryotic, for example mammalian, host cell expression systems mayalso be used. Suitable mammalian host cells include CHO, myeloma orhybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell containing a vector of the present invention underconditions suitable for leading to expression of protein from DNAencoding the antibody molecule of the present invention, and isolatingthe antibody molecule.

For production of products comprising both heavy and light chains, thecell line may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.In one example the cell line may be transfected with three vectors, eachencoding a polypeptide chain of an antibody molecule of the presentinvention. In one example the cell line is transfected with threevectors each one encoding a different polypeptide selected from a) apolypeptide chain of formula (I):

(Vxx)_(n)Vx-Cx-X-V₁;

b) a polypeptide chain of formula (II):

(Vyy)_(n)Vy-C_(y)

and c) a polypeptide of formula (III):

V₂

whereinVx represents a variable domain,Vxx represents a variable domain,Cx represents a constant region domain,X represents a linker,V₁ represents a variable domain,Vy represents a variable domain,Vyy represents a variable domain,Cy represents a constant region domain,V₂ represents a variable domain,n independently represents 0 or 1,

It will be appreciated that the ratio of each vector transfected intothe host cell may be varied in order to optimise expression of themulti-specific antibody product. In one embodiment the ratio of vectorsis 1:1:1. It will be appreciated that skilled person is able to find anoptimal ratio by routine testing of protein expression levels followingtransfection.

It will also be appreciated that where two or more, in particular threeof more, of the polypeptide components are encoded by a polynucleotidein a single vector the relative expression of each polypeptide componentcan be varied by utilising different promoters for each polynucleotideencoding a polypeptide component of the present invention.

In one embodiment the vector comprises a single polynucleotide sequenceencoding all three polypeptide chains of the multispecific antibodymolecule of the present invention under the control of a singlepromoter.

In one embodiment the vector comprises a single polynucleotide sequenceencoding all three polypeptide chains of the multispecific antibodymolecule of the present invention wherein each polynucleotide sequenceencoding each polypeptide chain is under the control of a differentpromoter.

The antibodies and fragments according to the present disclosure areexpressed at good levels from host cells. Thus the properties of theantibodies and/or fragments appear to be optimised and conducive tocommercial processing.

The antibodies of the present invention are useful in the treatmentand/or prophylaxis of a pathological condition.

The present invention also provides a pharmaceutical or diagnosticcomposition comprising an antibody molecule of the present invention incombination with one or more of a pharmaceutically acceptable excipient,diluent or carrier. Accordingly, provided is the use of an antibody ofthe invention for use in treatment and for the manufacture of amedicament.

The composition will usually be supplied as part of a sterile,pharmaceutical composition that will normally include a pharmaceuticallyacceptable carrier. A pharmaceutical composition of the presentinvention may additionally comprise a pharmaceutically-acceptableadjuvant.

The present invention also provides a process for preparation of apharmaceutical or diagnostic composition comprising adding and mixingthe antibody molecule of the present invention together with one or moreof a pharmaceutically acceptable excipient, diluent or carrier.

The antibody molecule may be the sole active ingredient in thepharmaceutical or diagnostic composition or may be accompanied by otheractive ingredients including other antibody ingredients, for exampleanti-TNF, anti-IL-1β, anti-T cell, anti-IFNγ or anti-LPS antibodies, ornon-antibody ingredients such as xanthines. Other suitable activeingredients include antibodies capable of inducing tolerance, forexample, anti-CD3 or anti-CD4 antibodies.

In a further embodiment the antibody, fragment or composition accordingto the disclosure is employed in combination with a furtherpharmaceutically active agent, for example a corticosteroid (such asfluticasonoe propionate) and/or a beta-2-agonist (such as salbutamol,salmeterol or formoterol) or inhibitors of cell growth and proliferation(such as rapamycin, cyclophosphmide, methotrexate) or alternatively aCD28 and/or CD40 inhibitor. In one embodiment the inhibitor is a smallmolecule. In another embodiment the inhibitor is an antibody specific tothe target.

The pharmaceutical compositions suitably comprise a therapeuticallyeffective amount of the antibody of the invention. The term“therapeutically effective amount” as used herein refers to an amount ofa therapeutic agent needed to treat, ameliorate or prevent a targeteddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. For any antibody, the therapeutically effectiveamount can be estimated initially either in cell culture assays or inanimal models, usually in rodents, rabbits, dogs, pigs or primates. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

The precise therapeutically effective amount for a human subject willdepend upon the severity of the disease state, the general health of thesubject, the age, weight and gender of the subject, diet, time andfrequency of administration, drug combination(s), reaction sensitivitiesand tolerance/response to therapy. This amount can be determined byroutine experimentation and is within the judgement of the clinician.Generally, a therapeutically effective amount will be from 0.01 mg/kg to50 mg/kg, for example 0.1 mg/kg to 20 mg/kg. Alternatively, the dose maybe 1 to 500 mg per day such as 10 to 100, 200, 300 or 400 mg per day.Pharmaceutical compositions may be conveniently presented in unit doseforms containing a predetermined amount of an active agent of theinvention.

Compositions may be administered individually to a patient or may beadministered in combination (e.g. simultaneously, sequentially orseparately) with other agents, drugs or hormones.

The dose at which the antibody molecule of the present invention isadministered depends on the nature of the condition to be treated, theextent of the inflammation present and on whether the antibody moleculeis being used prophylactically or to treat an existing condition.

The frequency of dose will depend on the half-life of the antibodymolecule and the duration of its effect. If the antibody molecule has ashort half-life (e.g. 2 to 10 hours) it may be necessary to give one ormore doses per day. Alternatively, if the antibody molecule has a longhalf-life (e.g. 2 to 15 days) it may only be necessary to give a dosageonce per day, once per week or even once every 1 or 2 months.

The pharmaceutically acceptable carrier should not itself induce theproduction of antibodies harmful to the individual receiving thecomposition and should not be toxic. Suitable carriers may be large,slowly metabolised macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

Suitable forms for administration include forms suitable for parenteraladministration, e.g. by injection or infusion, for example by bolusinjection or continuous infusion. Where the product is for injection orinfusion, it may take the form of a suspension, solution or emulsion inan oily or aqueous vehicle and it may contain formulatory agents, suchas suspending, preservative, stabilising and/or dispersing agents.Alternatively, the antibody molecule may be in dry form, forreconstitution before use with an appropriate sterile liquid.

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals.However, in one or more embodiments the compositions are adapted foradministration to human subjects.

In one embodiment, in formulations according to the present disclosure,the pH of the final formulation is not similar to the value of theisoelectric point of the antibody or fragment, for if the pH of theformulation is 7 then a pI of from 8-9 or above may be appropriate.Whilst not wishing to be bound by theory it is thought that this mayultimately provide a final formulation with improved stability, forexample the antibody or fragment remains in solution.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, transcutaneous (for example, seeWO98/20734), subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, intravaginal or rectal routes. Hyposprays may alsobe used to administer the pharmaceutical compositions of the invention.Typically, the therapeutic compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. Preferably the antibody molecules of the presentinvention are administered subcutaneously, by inhalation or topically.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a specific tissue ofinterest. Dosage treatment may be a single dose schedule or a multipledose schedule.

It will be appreciated that the active ingredient in the compositionwill be an antibody molecule. As such, it will be susceptible todegradation in the gastrointestinal tract. Thus, if the composition isto be administered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Remington's Pharmaceutical Sciences (Mack PublishingCompany, N.J. 1991).

In one embodiment the formulation is provided as a formulation fortopical administrations including inhalation.

Suitable inhalable preparations include inhalable powders, meteringaerosols containing propellant gases or inhalable solutions free frompropellant gases (such as nebulisable solutions or suspensions).Inhalable powders according to the disclosure containing the activesubstance may consist solely of the abovementioned active substances orof a mixture of the above mentioned active substances withphysiologically acceptable excipient.

These inhalable powders may include monosaccharides (e.g. glucose orarabinose), disaccharides (e.g. lactose, saccharose, maltose), oligo-and polysaccharides (e.g. dextranes), polyalcohols (e.g. sorbitol,mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) ormixtures of these with one another. Mono- or disaccharides are suitablyused, the use of lactose or glucose, particularly but not exclusively inthe form of their hydrates.

Particles for deposition in the lung require a particle size less than10 microns, such as 1-9 microns for example from 0.1 to 5 μm, inparticular from 1 to 5 μm. The particle size of the active (such as theantibody or fragment is of primary importance).

The propellent gases which can be used to prepare the inhalable aerosolsare known in the art. Suitable propellent gases are selected from amonghydrocarbons such as n-propane, n-butane or isobutane andhalohydrocarbons such as chlorinated and/or fluorinated derivatives ofmethane, ethane, propane, butane, cyclopropane or cyclobutane. Theabovementioned propellent gases may be used on their own or in mixturesthereof.

Particularly suitable propellent gases are halogenated alkanederivatives selected from among TG 11, TG 12, TG 134a and TG227. Of theabovementioned halogenated hydrocarbons, TG134a(1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane)and mixtures thereof are particularly suitable.

The propellent-gas-containing inhalable aerosols may also contain otheringredients such as cosolvents, stabilisers, surface-active agents(surfactants), antioxidants, lubricants and means for adjusting the pH.All these ingredients are known in the art.

The propellant-gas-containing inhalable aerosols according to theinvention may contain up to 5% by weight of active substance. Aerosolsaccording to the invention contain, for example, 0.002 to 5% by weight,0.01 to 3% by weight, 0.015 to 2% by weight, 0.1 to 2% by weight, 0.5 to2% by weight or 0.5 to 1% by weight of active.

Alternatively topical administrations to the lung may also be byadministration of a liquid solution or suspension formulation, forexample employing a device such as a nebulizer, for example, a nebulizerconnected to a compressor (e.g., the Pari LC-Jet Plus® nebulizerconnected to a Pari Master® compressor manufactured by Pari RespiratoryEquipment, Inc., Richmond, Va.).

In one embodiment the formulation is provided as discrete ampoulescontaining a unit dose for delivery by nebulisation.

In one embodiment the antibody is supplied in lyophilised form, forreconstitutions or alternatively as a suspension formulation.

The antibody of the invention can be delivered dispersed in a solvent,e.g., in the form of a solution or a suspension. It can be suspended inan appropriate physiological solution, e.g., physiological saline, apharmacologically acceptable solvent or a buffered solution. Bufferedsolutions known in the art may contain 0.05 mg to 0.15 mg disodiumedetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25 mg polysorbate, 0.25 mgto 0.30 mg anhydrous citric acid, and 0.45 mg to 0.55 mg sodium citrateper 1 ml of water so as to achieve a pH of about 4.0 to 5.0. Asmentioned supra a suspension can made, for example, from lyophilisedantibody.

The therapeutic suspensions or solution formulations can also containone or more excipients.

Excipients are well known in the art and include buffers (e.g., citratebuffer, phosphate buffer, acetate buffer and bicarbonate buffer), aminoacids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g.,serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol,and glycerol. Solutions or suspensions can be encapsulated in liposomesor biodegradable microspheres. The formulation will generally beprovided in a substantially sterile form employing sterile manufactureprocesses.

This may include production and sterilization by filtration of thebuffered solvent solution used for the formulation, aseptic suspensionof the antibody in the sterile buffered solvent solution, and dispensingof the formulation into sterile receptacles by methods familiar to thoseof ordinary skill in the art.

Nebulisable formulation according to the present disclosure may beprovided, for example, as single dose units (e.g., sealed plasticcontainers or vials) packed in foil envelopes. Each vial contains a unitdose in a volume, e.g., 2 ml, of solvent/solution buffer.

The antibodies of the present disclosure are thought to be suitable fordelivery via nebulisation. It is also envisaged that the antibody of thepresent invention may be administered by use of gene therapy. In orderto achieve this, DNA sequences encoding the heavy and light chains ofthe antibody molecule under the control of appropriate DNA componentsare introduced into a patient such that the antibody chains areexpressed from the DNA sequences and assembled in situ.

The pathological condition or disorder, may, for example be selectedfrom the group consisting of infections (viral, bacterial, fungal andparasitic), endotoxic shock associated with infection, arthritis such asrheumatoid arthritis, asthma such as severe asthma, chronic obstructivepulmonary disease (COPD), pelvic inflammatory disease, Alzheimer'sDisease, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, Peyronie's Disease, coeliac disease, gallbladder disease,Pilonidal disease, peritonitis, psoriasis, vasculitis, surgicaladhesions, stroke, Type I Diabetes, lyme disease, meningoencephalitis,autoimmune uveitis, immune mediated inflammatory disorders of thecentral and peripheral nervous system such as multiple sclerosis, lupus(such as systemic lupus erythematosus) and Guillain-Barr syndrome,Atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave'sdisease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere'sdisease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma,Wegener's granulomatosis, other autoimmune disorders, pancreatitis,trauma (surgery), graft-versus-host disease, transplant rejection, heartdisease including ischaemic diseases such as myocardial infarction aswell as atherosclerosis, intravascular coagulation, bone resorption,osteoporosis, osteoarthritis, periodontitis and hypochlorhydia.

The present invention also provides an antibody molecule according tothe present invention for use in the treatment or prophylaxis of pain,particularly pain associated with inflammation.

Thus there is provided an antibody according to the invention for use intreatment and methods of treatment employing same.

In one embodiment there is provided a process for purifiying an antibody(in particular an antibody or fragment according to the invention).

In one embodiment there is provided a process for purifiying an antibody(in particular an antibody or fragment according to the invention)comprising the steps: performing anion exchange chromatography innon-binding mode such that the impurities are retained on the column andthe antibody is maintained in the unbound fraction. The step may, forexample be performed at a pH about 6-8.

The process may further comprise an intial capture step employing cationexchange chromatography, performed for example at a pH of about 4 to 5.

The process may further comprise of additional chromatography step(s) toensure product and process related impurities are appropriately resolvedfrom the product stream.

The purification process may also comprise of one or moreultra-filtration steps, such as a concentration and diafiltration step.

Purified form as used supra is intended to refer to at least 90% purity,such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w/w or more pure.

Substantially free of endotoxin is generally intended to refer to anendotoxin content of 1 EU per mg antibody product or less such as 0.5 or0.1 EU per mg product.

Substantially free of host cell protein or DNA is generally intended torefer to host cell protein and/or DNA content 400 μg per mg of antibodyproduct or less such as 100 μg per mg or less, in particular 20 μg permg, as appropriate.

The antibody molecule of the present invention may also be used indiagnosis, for example in the in vivo diagnosis and imaging of diseasestates involving OX40.

Comprising in the context of the present specification is intended tomeaning including.

Where technically appropriate embodiments of the invention may becombined.

Embodiments are described herein as comprising certainfeatures/elements. The disclosure also extends to separate embodimentsconsisting or consisting essentially of said features/elements.

The present invention is further described by way of illustration onlyin the following examples, which refer to the accompanying Figures, inwhich:

EXAMPLES Example 1: Single Linker Fab-dsFv Construction of Single LinkerA26Fab-645dsFv, A26Fab-645dsFv and A26Fab-645dsscFv Plasmids forExpression in Mammalian Cells

A26Fab fusion proteins for the expression of single linkerA26Fab-645dsFv and A26Fab-645dsFv, see FIG. 1, were constructed byfusing 645vL to the C-terminus of the Km3 allotype human kappa constantregion of the A26 light chain using the flexible linker SGGGGSGGGGSGGGGS(SEQ ID NO: 103), or by fusing 645vH to the C-terminus of the, γ1isotype human gamma-1 CH1 constant region of the A26 heavy chain usingthe flexible linker SGGGGSGGGGTGGGGS (SEQ ID NO: 78). In addition pointmutations were introduced into the DNA sequences at selected residues inthe framework region of both 645vL and 645vH. The mutations (heavy chainG44C and light chain G100C) were introduced to create an interchaindisulphide bond between the heavy and light chains of the 645Fv. A26Fabfusion proteins for the expression of A26Fab-645dsscFv, see FIG. 1, wereconstructed as follows. A single chain Fv (scFv) was constructed bylinking the N-terminus of 645vL to the C-terminus of 645vH via theflexible linker GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 79). Point mutationswere introduced into the DNA sequence at framework residues G100C in645vL and G44C in 645vH to make the disulphide linked scFv (dsscFv). The645dsscFv was then fused to the C-terminus of either the Km3 allotypehuman kappa constant region of the A26 light chain using the flexiblelinker SGGGGSGGGGS (SEQ ID NO: 104), or to the yl isotype human gamma-1CH1 constant region of the A26 heavy chain using the flexible linkerSGGGGTGGGGS (SEQ ID NO: 80). The A26Fab light chain-645dsvL, A26Fabheavy chain-645dsvH, A26Fab light chain, A26Fab heavy chain, 645dsvLfree domain, 645dsvH free domain, A26Fab light chain-645dsscFv andA26Fab heavy chain-645dsscFv were manufactured chemically andindividually cloned into mammalian expression vectors under the controlof the HCMV-MIE promoter and SV40E polyA sequence.

HEK293 Expression of Single Linker A26Fab-645dsFv, A26Fab-645dsFv andA26Fab-645dsscFv

HEK293 cells were transfected with the relevant plasmids usingInvitrogen's 293fectin transfection reagent according to themanufacturer's instructions. Plasmids were mixed as follows to expressthe different constructs; for A26Fab-645dsFv, the plasmids were A26FabHeavy-(S, 3×G4S/T)-645dsvH and A26Fab Light-(S, 3×G4S)-645dsvL; forSingle linker A26Fab-645dsFv (LC-vL linked), the plasmids were A26FabHeavy, 645dsvH and A26Fab Light-(S, 3×G4S)-645dsvL; for Single linkerA26Fab-645dsFv (HC-vH linked), the plasmids were A26Fab Heavy-(S,3×G4S/T)-645dsvH, A26Fab Light and 645dsvL; for A26Fab-645dsscFv(HC-scFv), the plasmids were A26Fab Heavy-(S,2×G4S/T)-645dsscFv(vH-4×G4S-vL) and A26Fab Light; and forA26Fab-645dsscFv (LC-scFv), the plasmids were A26Fab Heavy and A26FabLight-(S, 2×G4S)-645dsscFv(vH-4×G4S-vL). In addition the ratio of theplasmids used for the transfections also varied, for the 2 plasmidcombinations the ratio was 1:1, whereas for the 3 plasmid combinationsseveral different ratios were tested. A total of 50 μg of plasmid DNAwas incubated with 125 μl 293fectin+4.25 ml Optimem media for 20 mins atRT. The mixture was then added to 50 ml of HEK293 cells in suspension at1 25×10⁶ cells/ml and incubated with shaking at 37° C. Supernatants wereharvested on day 7 by centrifugation at 1500 g to remove cells and thesupernatant passed through a 0.224 μm filter. Expression level wasdetermined by Protein-G HPLC.

The level of expression of all the constructs was comparable, see table4, covering the range 3-15 g/ml. The Fab-dsFv expressed at 13-14 μg/ml,the Fab-dsscFv's expressed at 7-15 g/ml and the single linker Fab-dsFv'sexpressed at 3-13 μg/ml. There have been reports in the literature thatthe expression of Fv regions that lack either a linker between the vLand vH or a dimerisation motif to bring the vL and vH together havesubstantially lower expression levels than linked Fv's. This is notobserved in this data where there is no significant difference observedbetween the best expression of each type of construct.

TABLE 4 Expression level Construct (μg/ml) A26Fab-645dsFv 13.2-14.2A26Fab-645dsscFv (LC-scFv) 14.0-15.2 A26Fab-645dsscFv (HC-scFv) 6.6-7.1single linker A26Fab-645dsFv (LC-vL linked)(ratio 1:1:1 LC-vL:HC:vH)5.1-6.9 single linker A26Fab-645dsFv (LC-vL linked) (ratio 1:1:2LC-vL:HC:vH) 3.3-3.5 single linker A26Fab-645dsFv (LC-vL linked) (ratio2:1:2 LC-vL:HC:vH) 4.2-4.4 single linker A26Fab-645dsFv (LC-vL linked)(ratio 1:2:2 LC-vL:HC:vH) 4.2-4.3 single linker A26Fab-645dsFv (HC-vHlinked) (ratio 1:1:1 HC-vH:LC:vL)  6.8-12.6 single linker A26Fab-645dsFv(HC-vH linked) (ratio 1:1:2 HC-vH:LC:vL) 7.8-8.1 single linkerA26Fab-645dsFv (HC-vH linked) (ratio 2:1:2 HC-vH:LC:vL) 7.6-8.2BIAcore Analysis of HEK293 Expressed Single Linker A26Fab-645dsFv,A26Fab-645dsFv and A26Fab-645dsscFv

Binding affinities and kinetic parameters for the interactions ofFab-dsFv, Fab-dsscFv and single linker Fab-dsFv constructs weredetermined by surface plasmon resonance (SPR) conducted on a BIAcoreT100 using CM5 sensor chips and HBS-EP (10 mM HEPES (pH7.4), 150 mMNaCl, 3 mM EDTA, 0.05% v/v surfactant P20) running buffer. Single linkerFab-dsFv samples were captured to the sensor chip surface using either ahuman F(ab′)₂-specific goat Fab (Jackson ImmunoResearch, 109-006-097) oran in-house generated anti human CH1 monoclonal antibody. Covalentimmobilisation of the capture antibody was achieved by standard aminecoupling chemistry.

Each assay cycle consisted of firstly capturing the Fab-dsFv, Fab-dsscFvor single linker Fab-dsFv construct using a 1 min injection, before anassociation phase consisting of a 3 min injection of antigen, afterwhich dissociation was monitored for 5 min. After each cycle, thecapture surface was regenerated with 2×1 min injections of 40 mM HClfollowed by 30 s of 5 mM NaOH. The flow rates used were 10 l/min forcapture, 30 l/min for association and dissociation phases, and 10 l/minfor regeneration.

For kinetic assays, either a titration of human serum albumin 50-6.25nM, or a single concentration of OX40 of 25 nM was performed. A blankflow-cell was used for reference subtraction and buffer-blank injectionswere included to subtract instrument noise and drift.

Kinetic parameters were determined by simultaneous global-fitting of theresulting sensorgrams to a standard 1:1 binding model using BIAcore T100Evaluation software.

The on rates, off rates and affinities of all the samples are similarfor both antigens, human serum albumin (HSA) and human OX40, see table5. Therefore the presence and position of the different linkers in thedifferent constructs does not have a significant effect on the affinityof either variable region for its antigen.

TABLE 5 Sample Antigen ka (1/Ms) kd (1/s) KD (nM) Antigen ka (1/Ms) kd(1/s) KD (pM) A26Fab-645dsFv HSA 7.51E+04 1.51E−04 2.01 OX40 1.70E+051.53E−05 90 (start) A26Fab-645dsscFv HSA 1.83E+05 2.40E−04 1.31 OX401.66E+05 2.34E−05 141 (HC-scFv) A26Fab-645dsscFv HSA 1.72E+05 2.22E−041.29 OX40 1.78E+05 1.82E−05 102 (LC-scFv) Single linker A26Fab-645dsFv(LC-vL) HSA 7.40E+04 2.35E−04 3.17 OX40 2.29E+05 2.64E−05 115 (ratio1:1:1 LC-vL:HC:vH) Single linker A26Fab-645dsFv (LC-vL) HSA 1.13E+052.62E−04 2.31 OX40 1.84E+05 1.80E−05 98 (ratio 1:1:2 LC-vL:HC:vH) Singlelinker A26Fab-645dsFv (LC-vL) HSA 1.26E+05 1.88E−04 1.49 OX40 1.70E+051.69E−05 99 (ratio 2:1:2 LC-vL:HC:vH) Single linker A26Fab-645dsFv(LC-vL) HSA 9.10E+04 2.23E−04 2.46 OX40 1.54E+05 1.70E−05 110 (ratio1:1:1 LC-vL:HC:vH) Single linker A26Fab-645dsFv (HC-vH) HSA 2.09E+052.09E−04 1.00 OX40 1.73E+05 3.51E−05 203 (Ratio 1:1:1 HC-vH:LC:vL)Single linker A26Fab-645dsFv (HC-vH) HSA 2.11E+05 2.34E−04 1.11 OX401.92E+05 1.00E−05 52 (Ratio 1:1:2 HC-vH:LC:vL) Single linkerA26Fab-645dsFv (HC-vH) HSA 1.97E+05 2.07E−04 1.05 OX40 1.94E+05 1.97E−05101 (Ratio 2:1:2 HC-vH:LC:vL) A26Fab-645dsFv HSA 6.82E+04 2.12E−04 3.11OX40 1.93E+05 1.98E−05 102 (end)

Protein-G Purification of HEK293 Expressed Single Linker A26Fab-645dsFv,A26Fab-645dsFv and A26Fab-645dsscFv

The ˜50 ml HEK293 supernatants were concentrated ˜25 fold to ˜2 ml using10 kDa molecular weight cut off centrifugation concentrators. Theconcentrated supernatants were applied to a 1 ml HiTrap Protein-G FFcolumn (GE Healthcare) equilibrated in 20 mM phosphate, 40 mM NaClpH7.4. The column was washed with 20 mM phosphate, 40 mM NaCl pH7.4 andthe bound material eluted with 0.1M glycine/HCl pH2.7. The elution peakwas collected and pH adjusted to ˜pH7 with 2M Tris/HCl pH8.5. The pHadjusted elutions were concentrated and buffer exchanged into PBS pH7.4using 10 kDa molecular weight cut off centrifugation concentrators.

SDS-PAGE Analysis of Protein-G Purified, HEK293 Expressed, Single LinkerA26Fab-645dsFv, A26Fab-645dsFv and A26Fab-645dsscFv

Samples were diluted with water where required and then to 26 μl wasadded 10 μL 4×Bis-Tris LDS sample buffer and 4 μL of 10× reducing agentfor reduced samples. The samples were vortex mixed, incubated at 100° C.for 3 minutes, cooled and centrifuged at 12500 rpm for 30 seconds. Theprepared samples were loaded on to a 4-20% acrylamine Tris/Glycine SDSgel and run for 110 minutes at 125V, constant voltage. The gels werestained with Coomassie Blue protein stain, see FIG. 2.

The reducing SDS-PAGE gel has banding patterns in terms of bothmigration position and staining intensity that is constant with all theconstructs being expressed correctly. For Fab-dsFv, lane 2, there shouldbe 2 bands at ˜36 and ˜37 kDa with roughly equivalent staining. ForFab-dsscFv (LC-scFv), lane 3, there should be 2 bands at ˜51 and ˜23 kDawith roughly twice the staining in the upper band. For Fab-dsscFv(HC-scFv), lane 4, there should be 2 bands at ˜50 and ˜26 kDa withroughly twice the staining in the upper band. For single linker Fab-dsFv(LC-vL), lanes 6-9, there should be 3 bands at ˜36, ˜23 and ˜13 kDa withstaining roughly in the ratio 3:2:1 upper to lower band. For singlelinker Fab-dsFv (HC-vH), lanes 10-12, there should be 3 bands at ˜37,˜26 and ˜12 kDa with staining roughly in the ratio 3:2:1 upper to lowerband.

G3000 SEC-HPLC Analysis of Protein-G Purified, HEK293 Expressed, SingleLinker A26Fab-645dsFv, A26Fab-645dsFv and A26Fab-645dsscFv

50 μg samples were injected onto a TSK Gel G3000SWXL, 7.8×300 mm, column(Tosoh) and developed with an isocratic gradient of 200 mM phosphatepH7.0 at 1 ml/min. Signal detection was by absorbance at 280 nm, seeFIG. 3. After Protein-G purification A26Fab-645dsFv is ˜45% monomer,A26Fab-645dsscFv's have slightly more monomer in the range 55-60%,whereas the single linker A26Fab-645dsFv's are all in excess of 80%monomer with some being 100% monomer.

Example 2 Construction of Single Linker A26Fab-645dsFv andA26Fab-648dsFv Triple Gene Plasmids for Expression in Mammalian Cells

The triple gene plasmids were constructed by first generating anintermediate double gene vector from the single gene components of thesingle linker Fab-dsFv formats as described in example 1/FIG. 1. Thegene fragment encoding the expression of the heavy chain which includesthe hCMV-MIE promoter, the heavy chain and SV40 polyA region, wassub-cloned downstream of the light chain gene in a mammalian expressionvector. The heavy chain is either a A26 Fab heavy chain or the A26 Fabheavy linked to a 645dsvH or 648dsvH via a linker (S, 3×G₄S), and thelight chain is either a A26 Fab light linked to a 645dsvL or 648dsvL viaa linker (S, 3×G₄S) or a A26 Fab light chain, respectively. Thisgenerated the intermediate double gene plasmid for each format. Toconstruct the triple gene plasmid, the fragment encoding the expressionof the free cognate v region (645dsvL, 648dsvL, 645dsvH or 648dsvH), wassubsequently sub-cloned at a unique restriction site downstream of theheavy chain gene in the intermediate vector. For future stable cell linegeneration, a mammalian selection marker was finally sub-cloned into theexpression plasmids. This provided a set of plasmids that contained therelevant genes for single linker Fab-dsFv expression at equal generatios, with or without a mammalian selection marker. These plasmidswill be used for initial assessment in a transient mammalian expressionsystem for comparison with % monomer (FIG. 3) of single linker Fab-dsFvsexpressed from single gene plasmids.

Transient Expression of Single Linker A26Fab-645dsFv and A26Fab-648dsFvfrom Triple Gene Plasmids in CHO Cells

CHO cells were grown in CD CHO media supplemented with 1×L-glutaMAX(Life Technologies) to exponential phase with >99% viability. The cellswere prepared by washing in Earle's balanced salt solution (LifeTechnologies) and plasmid DNA was electroporated into the CHO cellsaccording to in-house recommendations. The transfected cells weretransferred to CD CHO medium supplemented with 1×L-glutaMAX and 1×anti-mycotic solution (Life Technologies) and incubated in an orbitalshaker for 24 h at 37° C., 8% CO₂, and shaking at 140 rpm. Followingincubation, or when the cultures had reached a viable cell density of atleast 2×10⁶ cells ml⁻¹, the temperature was decreased to 32° C.Subsequently after 72 h post-transfection, 3 mM sodium butyrate (SigmaAldrich) was added and the cultures were re-incubated for a further 11days at 32° C., with 8% CO₂ and shaking at 140 rpm. The supernatant washarvested by centrifugation and successively filtered through 0.45 μMand 0.22 μM sterile filters. Expression titres were quantified byprotein G HPLC against a Fab fragment standard.

The level of expression from triple gene plasmids was dependent on thepresence of the mammalian selection marker, see FIG. 5. Expressiontitres were higher amongst single linker A26 Fab-dsFv proteins expressedfrom plasmids without a mammalian selection marker whereas lower butcomparable levels were obtained if expressed from plasmids with themammalian selection marker, as would be expected due to the metabolicburden exacted by expression of an extra gene. Additionally, higherexpression titres were observed for proteins that contained an A26 Fablight linked to a 645dsvL or 648dsvL chain.

Protein G Purification of Single Linker A26Fab-645dsFv andA26Fab-648dsFv Expressed from Triple Gene Plasmids in CHO Cells

The 200 ml supernatants were concentrated by ˜20-fold using a 10 kDamolecular weight cut off centrifugation concentrators. The concentratedsupernatants were applied to a 1 ml HiTrap Protein-G FF column (GEHealthcare) equilibrated in 20 mM phosphate, 40 mM NaCl pH7.4. Thecolumn was washed with 20 mM phosphate, 40 mM NaCl pH7.4 and the boundmaterial eluted with 0.1M glycine/HCl pH2.7. The elution peak wascollected and the pH adjusted to ˜pH7 with Tris/HCl pH8.5. The pHadjusted elutions were concentrated and buffer exchanged into PBS pH7.4using 10 kDa molecular weight cut off centrifugation concentrators.Protein concentrations were estimated spectrophotometrically at A₂₈₀.

SDS-PAGE Analysis of Protein-G Purified Single Linker A26Fab-645dsFv andA26Fab-648dsFv Expressed from Triple Gene Plasmids in CHO Cells

Protein samples were diluted in PBS where required. To 8.25 μl of thesubsequent sample, 3.75 μl of 4×Bis-Tris LDS sample buffer (LifeTechnologies), 1.5 μl of 100 mM N-ethylmaleimide and 1.5 μl of 10×reducing agent were added for reduced samples. The samples were vortexmixed, incubated at 100° C. for 3 minutes, cooled and centrifuged at13200 rpm for 30 seconds. The prepared samples were loaded on to a 4-20%acrylamide Tris/Glycine SDS gel (Life Technologies) and run inTris-glycine buffer for ˜150 minutes at 125V, constant voltage. AnSDS-PAGE protein standard, Seeblue2 (Life Technologies) was used as thestandard marker. The gels were stained with InstantBlue Coomassie blueprotein stain (Expedeon) and destained with distilled water, see FIG. 6.

The reducing SDS-PAGE gel has banding patterns in terms of bothmigration position and staining intensity that is constant with all theconstructs being expressed correctly. For all single linker Fab-dsFvthere should be 3 bands at ˜36-37 (i), ˜24-26 (ii) and ˜12-13 kDa (iii),with staining should be roughly in the ratio of 3:2:1 upper to lowerband.

G3000 SEC-HPLC of Protein-G purified single linker A26Fab-645dsFv andA26Fab-648dsFv expressed from triple gene plasmids in CHO cells

50 ul samples were injected into a TSK Gel G3000SWXL, 7.8×300 mm, column(Tosoh) and developed with an isocratic gradient of 200 mM phosphatepH7.0 at 1 ml/min. Signal detection was by absorbance at 280 nm, seeFIG. 7. All single linker A26Fab-645dsFvs expressed from triple geneplasmids, irrespective of the presence of a mammalian selection markeror expression level, achieve in excess of 90% monomer, with the majoritybeing 100% monomer. This data is in good agreement with the monomericsingle linker A26Fab-dsFvs expressed from single gene plasmids, FIG. 3.This also indicates that an equal gene ratio of the relevant genesencoding the format, as present in a triple gene plasmid configurationis optimal for highly monomeric single linker Fab-dsFv expression.

1. A multi-specific antibody molecule consisting of three polypeptides,(a) a polypeptide chain of formula (I):(Vxx)_(n)Vx-Cx-X-V₁, (b) a polypeptide chain of formula (II):(Vyy)_(n)Vy-C_(y), and (c) a polypeptide of formula (III):V₂, wherein: Vx represents a variable domain, Vxx represents a variabledomain, Cx represents a constant region consisting of C_(L) or CH₁, Xrepresents a linker, V₁ represents a variable domain, Vy represents avariable domain, Vyy represents a variable domain, Cy represents aconstant region consisting of C_(L) or CH₁, V₂ represents a variabledomain, n independently represents 0 or 1; wherein only one of Cx or Cyis CH₁; wherein the polypeptide chain of formula (I) and the polypeptidechain of formula (II) is aligned such that the constant regions Cx andCy are paired, the variable domains Vx and Vy are paired to form abinding domain, the variable domains V₁ and V₂ are paired to form abinding domain, and a disulphide bond is present between V₁ and V₂;wherein the variable domain pair V₁/V₂ are linked by a disulfide bondbetween two engineered cysteine residues, one in V₁ and one in V₂; andwherein the position of the pair of engineered cysteine residues isselected from the group consisting of VH37 and VL95, VH44 and VL100,VH44 and VL105, VH45 and VL87, VH100 and VL50, VH100b and VL49, VH98 andVL46, VH101 and VL46, VH105 and VL43, and VH106 and VL57.
 2. Themulti-specific antibody molecule of claim 1, wherein (a) in thepolypeptide chain of formula (I), (Vxx)nVx is V_(L), (b) in thepolypeptide chain of formula (II), (Vyy)nVy is V_(H), (c) V_(H)represents a heavy chain variable domain, and (d) V_(L) represents alight chain variable domain.
 3. The multi-specific antibody molecule ofclaim 1, wherein Cx is C_(L), V₁ represents a light chain variabledomain and V₂ represents a heavy chain variable domain.
 4. Themulti-specific antibody molecule of claim 1, wherein Cx is CH1, V₁represents a heavy chain variable domain and V₂ represents a light chainvariable domain.
 5. The multi-specific antibody molecule of claim 1,wherein the variable domain pair V₁/V₂ have specificity for a serumcarrier protein.
 6. The multi-specific antibody molecule of claim 5,wherein V₂ comprises the sequence given in SEQ ID NO:87 for CDRH-1, thesequence given in SEQ ID NO:88 for CDRH2 and the sequence given in SEQID NO:89 for CDRH-3 and V₁ comprises the sequence given in SEQ ID NO:90for CDRL-1, the sequence given in SEQ ID NO:91 for CDRL2 and thesequence given in SEQ ID NO:92 for CDRL-3.
 7. The multi-specificantibody molecule of claim 5, wherein V₂ comprises the sequence given inSEQ ID NO:93 for CDRH-1, the sequence given in SEQ ID NO:94 for CDRH2and the sequence given in SEQ ID NO:95 for CDRH-3 and V₁ comprises thesequence given in SEQ ID NO:96 for CDRL-1, the sequence given in SEQ IDNO:97 for CDRL2 and the sequence given in SEQ ID NO:98 for CDRL-3. 8.The multi-specific antibody molecule of claim 1, wherein X has thesequence given in SEQ ID NO:103.
 9. A polynucleotide, encoding themulti-specific antibody molecule of claim
 1. 10. A vector, comprisingthe polynucleotide of claim
 9. 11. A host cell, comprising thepolynucleotide of claim
 9. 12. A host cell, comprising three vectors,each vector comprising a polynucleotide encoding a different polypeptidechain of the multi-specific antibody molecule of claim
 1. 13. A process,comprising expressing a multi-specific molecule from the host cell ofclaim
 11. 14. The multi-specific antibody molecule of claim 1, wherein anatural disulfide bond is present between Cx and Cy.
 15. Themulti-specific antibody molecule of claim 1, wherein at least onebinding domain of the multi-specific antibody molecule is specific foran antigen that is an immunoglobulin, an interferon, a colonystimulating factor, a viral antigen, a member of the classical andalternative complement activation cascade, an FcγR, a complement pathwayprotein, an integrin, or an interleukin.
 16. The multi-specific antibodymolecule of claim 15, wherein the at least one binding domain isspecific for IgE.
 17. The multi-specific antibody molecule of claim 15,wherein the at least one binding domain is specific for an interferonthat is interferon α, interferon β, or interferon γ.
 18. Themulti-specific antibody molecule of claim 15, wherein the at least onebinding domain is specific for a colony stimulating factor that is G-CSFor GM-CSF.
 19. The multi-specific antibody molecule of claim 15, whereinthe at least one binding domain is specific for a viral antigen that isa respiratory syncytial virus or cytomegalovirus, influenza, EBV, HepA,B, or C antigen.
 20. The multi-specific antibody molecule of claim 15,wherein the at least one binding domain is specific for a member of theclassical and alternative complement activation cascade that is C2, C4,C3-convertase, C5, C6, C7, C8, or C9.
 21. The multi-specific antibodymolecule of claim 15, wherein the at least one binding domain isspecific for an FcγR that is FcγRI, FcγRII, or FcγRIII.
 22. Themulti-specific antibody molecule of claim 15, wherein the at least onebinding domain is specific for a complement pathway protein that is C1qor C3.
 23. The multi-specific antibody molecule of claim 15, wherein theat least one binding domain is specific for an integrin that is β1integrin, VLA-4, E-selectin, P selectin, or L-selectin.
 24. Themulti-specific antibody molecule of claim 15, wherein the at least onebinding domain is specific for an interleukin that is IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-8, IL-12, IL-16, IL-17, or IL-23.
 25. Themulti-specific antibody molecule of claim 15, wherein each bindingdomain is specific for an antigen independently selected from the groupconsisting of an integrin, CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD11b,CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CD45, CDW52, CD69, CD134(OX40), ICOS, BCMP7, CD137, CD27L, CDCP1, DPCR1, DPCR1, dudulin2,FLJ20584, FLJ40787, HEK2, KIAA0634, KIAA0659, KIAA1246, KIAA1455, LTBP2,LTK, MAL2, MRP2, nectin-like2, NKCC1, PTK7, RAIG1, TCAM1, SC6, BCMP101,BCMP84, BCMP11, DTD, carcinoembryonic antigen (CEA), human milk fatglobulin (HMFG1 and 2), MHC Class I and MHC Class II antigens, VEGF, aninterleukin, a viral antigen, an immunoglobulin, an interferon, tumournecrosis factor-α, tumor necrosis factor-β, a colony stimulating factor,a platelet derived growth factor, a bacterial cell surface antigen, abacterial toxins, a bioterrorism agent, a snake and spider venom andtoxin, OX40, histamine, C1q, opsonin, a member of the classical andalternative complement activation cascades, a FcγR, a complement pathwayprotein, a CD marker protein (Cluster of Differentiation marker, a serumcarrier protein, a circulating immunoglobulin molecule, and CD35/CR1.26. The multi-specific antibody molecule of claim 1, whereinC_(L)represents Ckappa or Clambda.
 27. A pharmaceutical composition,comprising the multi-specific antibody molecule of claim 1 and at leastone excipient.
 28. The pharmaceutical composition of claim 27, whereinat least 80% of the antibody molecules in the composition are inmonomeric form.
 29. A method of treatment, comprising administering amulti-specific antibody molecule of claim 1 to a subject in needthereof.